The present invention relates to a novel process for creating a robust, photocross-linkable collagen, and a process for creating a collagen and PEG-based hybrid biomaterial that allows for mechanical and biofunctional modulation using the application of light and a suitable photo-initiator.
Generally, collagen is a group of naturally occurring proteins found in animals, especially in the flesh and connective tissues of mammals. It is the main component of connective tissue, and is the most abundant protein in mammals, making up about 25% to 35% of the whole-body protein content. Collagen, in the form of elongated fibrils, is mostly found in fibrous tissues such as tendon, ligament and skin, and is also abundant in cornea, cartilage, bone, blood vessels, the gut, and intervertebral disc.
Type-I collagen is the major collagen of tendon and bone, but it is also the predominant in lung, skin, dentin, heart valves, fascia, scar tissue, cornea, and liver. Type I collagen is essential for the tensile strength of bone. It is the final amount and distribution of these collagen fibers that will determine the size, shape, and ultimate density of the bone.
Polyethylene glycol (“PEG”) has been used in medical implants and pharmaceuticals in a number of formulations for decades. In 1995, PEG was modified by adding reactive acrylate groups to the end of the PEG macromer to form PEG diacrylate, which was then photo-polymerized and used to form synthetic hydrogel matrices in which encapsulated cells could be grown.
Hydrogels are semi-solid structures comprising networks of water-insoluble polymers surrounded by water (Lee, et al., “Hydrogels for Tissue Engineering,” Chem. Rev., 101(7), 1869-1880 (2001)). They are attractive materials for use as tissue engineering scaffolds, particularly those made from materials that can polymerize in an aqueous environment that have potential to be injected into a defect or wound, and then polymerized to provide a stable matrix for cellular growth, remodeling, and regeneration into functional tissues (Nicodemus et al., “Cell Encapsulation in Biodegradable Hydrogels for Tissue Engineering Applications,” Tissue Eng. Part B-Rev., 14(2), 149-165 (2008); Bryant et al., “Encapsulating Chondrocytes in Degrading PEG Hydrogels With High Modulus: Engineering Gel Structural Changes to Facilitate Cartilaginous Tissue Production,” Biotech. Bioeng., 86(7), 747-55 (2004); Jen et al., “Review: Hydrogels for Cell Immobilization,” Biotech. Bioeng., 50(4), 357-364 (1996).) Natural hydrogels from proteins such as collagen, are both cytocompatible and highly biofunctional, but have somewhat constrained material properties and inherentability in composition due to their biological origin, making them more difficult to work with from an engineering viewpoint. (Cheung et al., “Mechanism of Cross-Linking of Proteins by Glutaraldehyde 0.3. Reaction with Collagen in Tissues,” Connect. Tiss. Res., 13(2), 109-115 (1985); Cen et al., “Collagen Tissue Engineering: Develop-ment of Novel Biomaterials and Applications,” Ped. Res., 63(5)492-496 (2008); Yang et al., “Mechanical Properties of Native and Cross-Linked Type I Collagen Fibrils,” Biophys. J., 94(6), 2204-2211 (2008).)
In recent years, the mechanical microenvironment has been elucidated as a potent modulator of cellular behavior and thus has been of great interest in designing scaffolds for tissue engineering. (Levenberg et al., “Cell-Scaffold Mechanical Interplay Within Engineered Tissue,”Sem. Cell Dev. Bio., 20(6), 656-664 (2009); Discher et al., Tissue Cells Feel and Respond to the Stiffness of Their Substrate, Science, 310(5751), 1139-1143 (2005); Rehfeldt et al., “Cell Responses to the Mechanochemical Microenvironment—Implications for Regenerative Medicine and Drug Delivery,” Adv. Drug Del. Rev., 59(13), 1329-1339 (2007).) In particular, stem cell-based tissue regeneration has shown scaffold mechanics to be of crucial importance in guiding and maintaining differentiation pathways. (O'Connor et al., “Review: Ex vivo Engineering of Living Tissues with Adult Stem Cells,” Tiss. Eng., 12(11), 3007-3019 (2006); Discher et al., “Matrix Elasticity Effects on Cardiomyocytes and Stem Cells: Similarities, Differences and Therapeutic Implications,” Biorheol., 45(1-2), 54 (2008); Discher et al., “Matrix Elasticity Directs Stem Cell Lineage Specification,” Biophys. J., 32a-32a (2007).) Synthetic scaffolds are increasingly popular, partly due to the ease with which their mechanical properties—as well as other characteristics—can be modulated. Natural materials such as collagen, while having the benefits of bio-activity, biodegradability, and innate adhesiveness, have been criticized for the limited control of their mechanical properties. (Lau et al., A Critical Review on Polymer-Based Bio-Engineered Materials for Scaffold Development,” Compos. Part B-Eng., 38(3), 291-300 (2007).)
Hybrid materials, which contain a mixture of biomaterials and synthetic components, are becoming popular as tissue engineering matrices due to the combination of their respective advantages. Several groups have used combinations of natural and synthetic materials to optimize and tailor the properties of tissue engineering scaffolds to the particular application. However, simply combining biomaterials with synthetics has limitations, due to the drawbacks of having both materials everywhere within the scaffold. In the case of PEG, this could prevent cell attachment.
Other approaches using hybrid materials involve using collagen as a base material, and admixing additional natural or synthetic components such as hyaluronic acid and polyethylene oxide to form interpenetrating networks. A major drawback to this system is again there is little control over where materials interact, and it may be hard to determine with which material cells might interface due to the presence of two independent matrices.
Previous attempts at modifying collagen's material properties have presented significant challenges. Although chemical cross-linking using glutaraldehyde provides significant increases in mechanical strength, it is highly cytotoxic. More cytocompatible cross-linking compounds, such as genipin, allow cross-linking in the presence of cells, although the degree of cross-linking is limited and localization of cross-linking is difficult due to diffusion of chemical agents through the hydrogel. Enzymes, such as transglutaminases, are non-cytotoxic but are prohibitively expensive and are also subject to uncontrolled diffusion. Other approaches, such as exposure to UV light are either cytotoxic, in the case of UVC, or minimally effective and slow, as with UVA exposure. Collagen has been reportedly directly crosslinked using UV light with riboflavin as a photosensitizing agent, although numerous tests have shown this method does not significantly change the mechanical properties, and is quite cytotoxic as well.
Photocrosslinking of collagen has been pursued in a variety of ways. UV irradiation in the presence of flavin mononucleotide produced only minimal changes in mechanical properties, and only when crosslinking was done prior to self-assembly. (Ibusuki et al., “Photochemically Cross-Linked Collagen Gels as Three-Dimensional Scaffolds for Tissue Engineering,” Tiss. Eng., 13(8), 1995-2001 (2007).) Collagen has also been modified via addition of photosensitive cinnamate groups, although the wavelength needed to crosslink is cytotoxic which prevents crosslinking in the presence of cells. (Dong et al., Photomediated Cros slinking of C6-Cinnamate Derivatized Type I Collagen,” Biomater., 26(18), 4041-4049 (2005).) Other approaches first modified collagen with either acrylate or methacrylate groups prior to photoinitiator-activated cross-linking. (Poshusta et al., “Photopolymerized Biomaterials for Application in the Temporo-mandibular Joint,” Cells Tiss. Orgs., 169(3), 272-278 (2001); Brinkman et al., “Photo-cross-linking of Type I Collagen Gels in the Presence of Smooth Muscle Cells: Mechanical Properties, Cell Viability and Function,” Biomacromols., 4(4), 890-895 (2003).) However, reaction conditions in these methods resulted in either unwanted gelation during reaction or partial denaturation of the collagen, which, while producing a useful photosensitive material, resulted in loss of the collagen to self-assemble into fibrils similar to native collagen.
More recently, there have been attempts made to modify collagen with photoactive groups such that light, in conjunction with a photoinitiator, might be used to significantly produce material changes spatially and in a cytocompatible manner. However, these approaches appear to have the common problem that the reaction conditions under which the collagen is modified are too harsh to preserve the complex tertiary structure, the result of which is that the collagen becomes partially denatured and is no longer able to spontaneously self-assemble.
Several groups have used collagen gels as matrices for stem and neural precursor cell-based therapies in central nervous system (“CNS”) injury models. While their results show that collagen gels are suitable for supporting both stem cell proliferation and differentiation into neural tissues, prior to the development of the instant invention these materials lacked the ability to produce localized, controlled heterogeneity, which may be necessary to completely regenerate damaged tissues and restore function to pre-injury levels.